Engineered <
em>
S. cerevisiae<
/em>
employing the xylose reductase pathway enables efficient xylose valorization to fuels and chemicals. However, toxicity of thermochemically pretreated biomass hydrolysate on <
em>
S. cerevisiae<
/em>
is one of the key technical challenges to upgrade biomass-derived sugars including xylose and glucose into high-value products. We investigated the effect of glycolaldehyde, one of the biomass-derived highly toxic aldehyde compounds, and its combinatorial inhibitory effect with other major fermentation inhibitors commonly found in plant hydrolysate such as methylglyoxal, 5-HMF, furfural, vanillin, and acetic acid on engineered xylose-fermenting <
em>
S. cerevisiae<
/em>
in xylose and/or glucose media. We elucidated that glycolaldehyde and methylglyoxal are the key inhibitory short-aliphatic aldehydes on engineered xylose-fermenting <
em>
S. cerevisiae<
/em>
in xylose-containing medium. Indeed, the degree of toxicity of these tested fermentation inhibitors varies with the sole carbon source of the medium. We demonstrate that genome integration of an extra copy of autologous <
em>
GRE2<
/em>
with its native promotor substantially improved the toxic tolerance of engineered xylose-fermenting <
em>
S. cerevisiae<
/em>
to major inhibitory compounds including glycolaldehyde in the xylose-containing medium, and xylose-rich, lignocellulosic hydrolysate derived from Miscanthus giganteus, and concurrently improved the ethanol fermentation profile. In conclusion, outcomes of this study will aid the development of next-generation robust <
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S. cerevisiae<
/em>
strains for efficient fermentation of hexose and pentose sugars found in biomass hydrolysate.